The present invention relates generally to the identification and isolation of novel chimpanzee erythropoietin polypeptides, nucleic acid molecules encoding those polypeptides and to the recombinant production of those polypeptides.
Erythropoiesis, the production of red blood cells, occurs continuously throughout the human life span to offset cell destruction. Erythropoiesis is a very precisely controlled physiological mechanism enabling sufficient numbers of red blood cells to be available in the blood for proper tissue oxygenation, but not so many that the cells would impede circulation. The formation of red blood cells occurs in the bone marrow and is under control of the hormone, erythropoietin.
Erythropoietin, an acidic glycoprotein is approximately 34,000 dalton molecular weight, may occur in three forms: alpha, beta and asialo. The alpha and beta forms different slightly in carbohydrate components have the same potency, biological activity and molecular weight. The asialo form is an alpha or beta form with the terminal carbohydrate (sialic acid) removed. Erythropoietin is present in a very low concentrations in plasma when the body is in a healthy state wherein tissues receive sufficient oxygenation from the existing number of erythrocytes. This normal low concentration is enough to stimulate replacement of red blood cells which are lost normally through aging.
The amount of erythropoietin in the circulation is increased under conditions of hypoxia when oxygen transport by blood cells in the circulation is reduced. Hypoxia may be caused by loss of large amounts of blood through hemorrhage, destruction of red blood cells by over-exposure to radiation, reduction in oxygen intake due to high altitudes or prolonged unconsciousness, or various forms of anemia. In response to tissues undergoing hypoxic stress, erythropoietin will increase red blood cell production by stimulating the conversion of primitive precursor cells in the bone marrow into proerythroblasts which subsequently mature, synthesize hemoglobin and are released into the circulation as red blood cells. When the number of red blood cells in circulation is greater than needed for normal tissue oxygen requirements, erythropoietin in circulation is decreased.
Because erythropoietin is essential in the process of red blood cell formation, the hormone has potential useful application in both the diagnosis and treatment of blood disorders characterized by low or defective red blood cell production. See, generally, Pennathur-Das, et al., Blood 63(5):1168-71 (1984) and Haddy, Am. Jour. Ped. Hematol. Oncol., 4:191-196 (1982) relating to erythropoietin in possible therapies for sickle cell disease, and Eschbach et al., J. Clin. Invest. 74(2):434-441 (1984), describing a therapeutic regimen for uremic sheep based on in vivo response to erythropoietin-rich plasma infusions and proposing a dosage of 10 U EOP/kg per day for 15-40 days as corrective of anemia of the type associated with chronic renal failure. See also, Krane, Henry Ford Hosp. Med. J., 31(3):177-181 (1983).
We describe herein the identification and characterization of a novel erythropoietin polypeptide derived from the chimpanzee, designated herein as xe2x80x9cCHEPOxe2x80x9d.
A cDNA clone has been identified that has homology to nucleic acid encoding human erythropoietin that encodes a novel chimpanzee erythropoietin polypeptide, designated in the present application as xe2x80x9cCHEPOxe2x80x9d.
In one embodiment, the invention provides an isolated nucleic acid molecule comprising a nucleotide sequence that encodes a CHEPO polypeptide.
In one aspect, the isolated nucleic acid molecule comprises a nucleotide sequence having at least about 80% nucleic acid sequence identity, alternatively at least about 81% nucleic acid sequence identity, alternatively at least about 82% nucleic acid sequence identity, alternatively at least about 83% nucleic acid sequence identity, alternatively at least about 84% nucleic acid sequence identity, alternatively at least about 85% nucleic acid sequence identity, alternatively at least about 86% nucleic acid sequence identity, alternatively at least about 87% nucleic acid sequence identity, alternatively at least about 88% nucleic acid sequence identity, alternatively at least about 89% nucleic acid sequence identity, alternatively at least about 90% nucleic acid sequence identity, alternatively at least about 91% nucleic acid sequence identity, alternatively at least about 92% nucleic acid sequence identity, alternatively at least about 93% nucleic acid sequence identity, alternatively at least about 94% nucleic acid sequence identity, alternatively at least about 95% nucleic acid sequence identity, alternatively at least about 96% nucleic acid sequence identity, alternatively at least about 97% nucleic acid sequence identity, alternatively at least about 98% nucleic acid sequence identity and alternatively at least about 99% nucleic acid sequence identity to (a) a DNA molecule encoding a CHEPO polypeptide having the sequence of amino acid residues from about 1 or about 28 to about 193, inclusive, of FIG. 3 (SEQ ID NOS:2 and 5), or (b) the complement of the DNA molecule of (a).
In another aspect, the isolated nucleic acid molecule comprises (a) a nucleotide sequence encoding a CHEPO polypeptide having the sequence of amino acid residues from about 1 or about 28 to about 193, inclusive, of FIG. 3 (SEQ ID NOS:2 and 5), or (b) the complement of the nucleotide sequence of (a).
In other aspects, the isolated nucleic acid molecule comprises a nucleotide sequence having at least about 80% nucleic acid sequence identity, alternatively at least about 81% nucleic acid sequence identity, alternatively at least about 82% nucleic acid sequence identity, alternatively at least about 83% nucleic acid sequence identity, alternatively at least about 84% nucleic acid sequence identity, alternatively at least about 85% nucleic acid sequence identity, alternatively at least about 86% nucleic acid sequence identity, alternatively at least about 87% nucleic acid sequence identity, alternatively at least about 88% nucleic acid sequence identity, alternatively at least about 89% nucleic acid sequence identity, alternatively at least about 90% nucleic acid sequence identity, alternatively at least about 91% nucleic acid sequence identity, alternatively at least about 92% nucleic acid sequence identity, alternatively at least about 93% nucleic acid sequence identity, alternatively at least about 94% nucleic acid sequence identity, alternatively at least about 95% nucleic acid sequence identity, alternatively at least about 96% nucleic acid sequence identity, alternatively at least about 97% nucleic acid sequence identity, alternatively at least about 98% nucleic acid sequence identity and alternatively at least about 99% nucleic acid sequence identity to (a) a DNA molecule having the sequence of nucleotides from about 1 or about 82 to about 579, inclusive, of FIG. 2 (SEQ ID NO:3), or (b) the complement of the DNA molecule of (a).
In another aspect, the isolated nucleic acid molecule comprises (a) the nucleotide sequence of from about 1 or about 82 to about 579, inclusive, of FIG. 2 (SEQ ID NO:3), or (b) the complement of the nucleotide sequence of (a).
In another aspect, the invention concerns an isolated nucleic acid molecule which encodes an active CHEPO polypeptide as defined below comprising a nucleotide sequence that hybridizes to the complement of a nucleic acid sequence that encodes amino acids 1 or about 28 to about 193, inclusive, of FIG. 3 (SEQ ID NOS:2 and 5). Preferably, hybridization occurs under stringent hybridization and wash conditions.
In yet another aspect, the invention concerns an isolated nucleic acid molecule which encodes an active CHEPO polypeptide as defined below comprising a nucleotide sequence that hybridizes to the complement of the nucleic acid sequence between about nucleotides 1 or about 82 and about 579, inclusive, of FIG. 2 (SEQ ID NO:3). Preferably, hybridization occurs under stringent hybridization and wash conditions.
In a further aspect, the invention concerns an isolated nucleic acid molecule which is produced by hybridizing a test DNA molecule under stringent conditions with (a) a DNA molecule encoding a CHEPO polypeptide having the sequence of amino acid residues from about 1 or about 28 to about 193, inclusive, of FIG. 3 (SEQ ID NOS:2 and 5), or (b) the complement of the DNA molecule of (a), and, if the test DNA molecule has at least about an 80% nucleic acid sequence identity, alternatively at least about 81% nucleic acid sequence identity, alternatively at least about 82% nucleic acid sequence identity, alternatively at least about 83% nucleic acid sequence identity, alternatively at least about 84% nucleic acid sequence identity, alternatively at least about 85% nucleic acid sequence identity, alternatively at least about 86% nucleic acid sequence identity, alternatively at least about 87% nucleic acid sequence identity, alternatively at least about 88% nucleic acid sequence identity, alternatively at least about 89% nucleic acid sequence identity, alternatively at least about 90% nucleic acid sequence identity, alternatively at least about 91% nucleic acid sequence identity, alternatively at least about 92% nucleic acid sequence identity, alternatively at least about 93% nucleic acid sequence identity, alternatively at least about 94% nucleic acid sequence identity, alternatively at least about 95% nucleic acid sequence identity, alternatively at least about 96% nucleic acid sequence identity, alternatively at least about 97% nucleic acid sequence identity, alternatively at least about 98% nucleic acid sequence identity and alternatively at least about 99% nucleic acid sequence identity to (a) or (b), and isolating the test DNA molecule.
In a specific aspect, the invention provides an isolated nucleic acid molecule comprising DNA encoding a CHEPO polypeptide without the N-terminal signal sequence and/or the initiating methionine, or is complementary to such encoding nucleic acid molecule. The signal peptide has been tentatively identified as extending from about amino acid position 1 to about amino acid position 27 in the sequence of FIG. 3 (SEQ ID NOS:2 and 5). It is noted, however, that the C-terminal boundary of the signal peptide may vary, but most likely by no more than about 5 amino acids on either side of the signal peptide C-terminal boundary as initially identified herein, wherein the C-terminal boundary of the signal peptide may be identified pursuant to criteria routinely employed in the art for identifying that type of amino acid sequence element (e.g., Nielsen et al., Prot. Eng. 10:1-6 (1997) and von Heinje et al., Nucl. Acids. Res. 14:4683-4690 (1986)). Moreover, it is also recognized that, in some cases, cleavage of a signal sequence from a secreted polypeptide is not entirely uniform, resulting in more than one secreted species. These polypeptides, and the polynucleotides encoding them, are contemplated by the present invention. As such, for purposes of the present application, the signal peptide of the CHEPO polypeptide shown in FIG. 3 (SEQ ID NOS:2 and 5) extends from amino acids 1 to X of FIG. 3 (SEQ ID NOS:2 and 5), wherein X is any amino acid from 23 to 32 of FIG. 3 (SEQ ID NOS:2 and 5). Therefore, mature forms of the CHEPO polypeptide which are encompassed by the present invention include those comprising amino acids X to 193 of FIG. 3 (SEQ ID NOS:2 and 5), wherein X is any amino acid from 23 to 32 of FIG. 3 (SEQ ID NOS:2 and 5) and variants thereof as described below. Isolated nucleic acid molecules encoding these polypeptides are also contemplated.
Another embodiment is directed to fragments of a CHEPO polypeptide coding sequence that may find use as, for example, hybridization probes or for encoding fragments of a CHEPO polypeptide that may optionally encode a polypeptide comprising a binding site for an anti-CHEPO antibody. Such nucleic acid fragments are usually at least about 20 nucleotides in length, alternatively at least about 30 nucleotides in length, alternatively at least about 40 nucleotides in length, alternatively at least about 50 nucleotides in length, alternatively at least about 60 nucleotides in length, alternatively at least about 70 nucleotides in length, alternatively at least about 80 nucleotides in length, alternatively at least about 90 nucleotides in length, alternatively at least about 100 nucleotides in length, alternatively at least about 110 nucleotides in length, alternatively at least about 120 nucleotides in length, alternatively at least about 130 nucleotides in length, alternatively at least about 140 nucleotides in length, alternatively at least about 150 nucleotides in length, alternatively at least about 160 nucleotides in length, alternatively at least about 170 nucleotides in length, alternatively at least about 180 nucleotides in length, alternatively at least about 190 nucleotides in length, alternatively at least about 200 nucleotides in length, alternatively at least about 250 nucleotides in length, alternatively at least about 300 nucleotides in length, alternatively at least about 350 nucleotides in length, alternatively at least about 400 nucleotides in length, alternatively at least about 450 nucleotides in length, alternatively at least about 500 nucleotides in length, alternatively at least about 600 nucleotides in length, alternatively at least about 700 nucleotides in length, alternatively at least about 800 nucleotides in length, alternatively at least about 900 nucleotides in length and alternatively at least about 1000 nucleotides in length, wherein in this context the term xe2x80x9caboutxe2x80x9d means the referenced nucleotide sequence length plus or minus 10% of that referenced length. In a preferred embodiment, the nucleotide sequence fragment is derived from any coding region of the nucleotide sequence shown in FIG. 1 (SEQ ID NO:1). It is noted that novel fragments of a CHEPO polypeptide-encoding nucleotide sequence may be determined in a routine manner by aligning the CHEPO polypeptide-encoding nucleotide sequence with other known nucleotide sequences using any of a number of well known sequence alignment programs and determining which CHEPO polypeptide-encoding nucleotide sequence fragment(s) are novel. All of such CHEPO polypeptide-encoding nucleotide sequences are contemplated herein and can be determined without undue experimentation. Also contemplated are the CHEPO polypeptide fragments encoded by these nucleotide molecule fragments, preferably those CHEPO polypeptide fragments that comprise a binding site for an anti-CHEPO antibody.
In another embodiment, the invention provides a vector comprising a nucleotide sequence encoding CHEPO or its variants. The vector may comprise any of the isolated nucleic acid molecules hereinabove identified.
A host cell comprising such a vector is also provided. By way of example, the host cells may be CHO cells, E. coli, or yeast. A process for producing CHEPO polypeptides is further provided and comprises culturing host cells under conditions suitable for expression of CHEPO and recovering CHEPO from the cell culture.
In another embodiment, the invention provides isolated CHEPO polypeptide encoded by any of the isolated nucleic acid sequences hereinabove identified.
In a specific aspect, the invention provides isolated native sequence CHEPO polypeptide, which in certain embodiments, includes an amino acid sequence comprising residues from about 1 or about 28 to about 193 of FIG. 3 (SEQ ID NOS:2 and 5).
In another aspect, the invention concerns an isolated CHEPO polypeptide, comprising an amino acid sequence having at least about 80% amino acid sequence identity, alternatively at least about 81% amino acid sequence identity, alternatively at least about 82% amino acid sequence identity, alternatively at least about 83% amino acid sequence identity, alternatively at least about 84% amino acid sequence identity, alternatively at least about 85% amino acid sequence identity, alternatively at least about 86% amino acid sequence identity, alternatively at least about 87% amino acid sequence identity, alternatively at least about 88% amino acid sequence identity, alternatively at least about 89% amino acid sequence identity, alternatively at least about 90% amino acid sequence identity, alternatively at least about 91% amino acid sequence identity, alternatively at least about 92% amino acid sequence identity, alternatively at least about 93% amino acid sequence identity, alternatively at least about 94% amino acid sequence identity, alternatively at least about 95% amino acid sequence identity, alternatively at least about 96% amino acid sequence identity, alternatively at least about 97% amino acid sequence identity, alternatively at least about 98% amino acid sequence identity and alternatively at least about 99% amino acid sequence identity to the sequence of amino acid residues from about 1 or about 28 to about 193, inclusive, of FIG. 3 (SEQ ID NOS:2 and 5).
In a specific aspect, the invention provides an isolated CHEPO polypeptide without the N-terminal signal sequence and/or the initiating methionine and is encoded by a nucleotide sequence that encodes such an amino acid sequence as hereinbefore described. Processes for producing the same are also herein described, wherein those processes comprise culturing a host cell comprising a vector which comprises the appropriate encoding nucleic acid molecule under conditions suitable for expression of the CHEPO polypeptide and recovering the CHEPO polypeptide from the cell culture.
In yet another aspect, the invention concerns an isolated CHEPO polypeptide, comprising the sequence of amino acid residues from about 1 or about 28 to about 193, inclusive, of FIG. 3 (SEQ ID NOS:2 and 5), or a fragment thereof which is biologically active or sufficient to provide a binding site for an anti-CHEPO antibody, wherein the identification of CHEPO polypeptide fragments that possess biological activity or provide a binding site for an anti-CHEPO antibody may be accomplished in a routine manner using techniques which are well known in the art. Preferably, the CHEPO fragment retains a qualitative biological activity of a native CHEPO polypeptide.
In a still further aspect, the invention provides a polypeptide produced by (i) hybridizing a test DNA molecule under stringent conditions with (a) a DNA molecule encoding a CHEPO polypeptide having the sequence of amino acid residues from about 1 or about 28 to about 193, inclusive, of FIG. 3 (SEQ ID NOS:2 and 5), or (b) the complement of the DNA molecule of (a), and if the test DNA molecule has at least about an 80% nucleic acid sequence identity, alternatively at least about 81% nucleic acid sequence identity, alternatively at least about 82% nucleic acid sequence identity, alternatively at least about 83% nucleic acid sequence identity, alternatively at least about 84% nucleic acid sequence identity, alternatively at least about 85% nucleic acid sequence identity, alternatively at least about 86% nucleic acid sequence identity, alternatively at least about 87% nucleic acid sequence identity, alternatively at least about 88% nucleic acid sequence identity, alternatively at least about 89% nucleic acid sequence identity, alternatively at least about 90% nucleic acid sequence identity, alternatively at least about 91% nucleic acid sequence identity, alternatively at least about 92% nucleic acid sequence identity, alternatively at least about 93% nucleic acid sequence identity, alternatively at least about 94% nucleic acid sequence identity, alternatively at least about 95% nucleic acid sequence identity, alternatively at least about 96% nucleic acid sequence identity, alternatively at least about 97% nucleic acid sequence identity, alternatively at least about 98% nucleic acid sequence identity and alternatively at least about 99% nucleic acid sequence identity to (a) or (b), (ii) culturing a host cell comprising the test DNA molecule under conditions suitable for expression of the polypeptide, and (iii) recovering the polypeptide from the cell culture.
In another embodiment, the invention provides chimeric molecules comprising a CHEPO polypeptide fused to a heterologous polypeptide or amino acid sequence, wherein the CHEPO polypeptide may comprise any CHEPO polypeptide, variant or fragment thereof as hereinbefore described. An example of such a chimeric molecule comprises a CHEPO polypeptide fused to an epitope tag sequence or a Fc region of an immunoglobulin.
In another embodiment, the invention provides an antibody as defined below which specifically binds to a CHEPO polypeptide as hereinbefore described. Optionally, the antibody is a monoclonal antibody, an antibody fragment or a single chain antibody.
In yet another embodiment, the invention concerns agonists and antagonists of a native CHEPO polypeptide as defined below. In a particular embodiment, the agonist or antagonist is an anti-CHEPO antibody or a small molecule.
In a further embodiment, the invention concerns a method of identifying agonists or antagonists to a CHEPO polypeptide which comprise contacting the CHEPO polypeptide with a candidate molecule and monitoring a biological activity mediated by said CHEPO polypeptide. Preferably, the CHEPO polypeptide is a native CHEPO polypeptide.
In a still further embodiment, the invention concerns a composition of matter comprising a CHEPO polypeptide, or an agonist or antagonist of a CHEPO polypeptide as herein described, or an anti-CHEPO antibody, in combination with a carrier. Optionally, the carrier is a pharmaceutically acceptable carrier.
Another embodiment of the present invention is directed to the use of a CHEPO polypeptide, or an agonist or antagonist thereof as herein described, or an anti-CHEPO antibody, for the preparation of a medicament useful in the treatment of a condition which is responsive to the CHEPO polypeptide, an agonist or antagonist thereof or an anti-CHEPO antibody.
Yet another embodiment of the present invention is directed to CHEPO polypeptides having altered glycosylation patterns in one or more regions of the polypeptide as compared to the native sequence CHEPO polypeptide, preferably in the region surrounding and/or including amino acid position 84 in the CHEPO amino acids sequence shown in FIG. 3 (SEQ ID NOS:2 and 5). In various embodiments, CHEPO variant polypeptides are prepared using well known techniques so as to create an N- or O-linked glycosylation site at or near amino acid position 84 in the CHEPO polypeptide sequence. For example, CHEPO polypeptides contemplated by the present invention include those where (a) amino acids 81-84 of the CHEPO amino acid sequence shown in FIG. 3 (SEQ ID NOS:2 and 5) (i.e., Met-Glu-Val-Arg; SEQ ID NO:6) are replaced by the amino acid sequence Asn-X-Ser-X (SEQ ID NO:7) or Asn-X-Thr-X (SEQ ID NO:8), where X is any amino acid except for Pro; (b) amino acids 82-85 of the CHEPO amino acid sequence shown in FIG. 3 (SEQ ID NOS:2 and 5) (i.e., Glu-Val-Arg-Gln; SEQ ID NO:9) are replaced by the amino acid sequence Asn-X-Ser-X (SEQ ID NO:7) or Asn-X-Thr-X (SEQ ID NO:8), where X is any amino acid except for Pro; (c) amino acids 83-86 of the CHEPO amino acid sequence shown in FIG. 3 (SEQ ID NOS:2 and 5) (i.e., Val-Arg-Gln-Gln; SEQ ID NO:10) are replaced by the amino acid sequence Asn-X-Ser-X (SEQ ID NO:7) or Asn-X-Thr-X (SEQ ID NO:8), where X is any amino acid except for Pro; or (d) amino acids 84-87 of the CHEPO amino acid sequence shown in FIG. 3 (SEQ ID NOS:2 and 5) (i.e., Arg-Gln-Gln-Ala; SEQ ID NO:11) are replaced by the amino acid sequence Asn-X-Ser-X (SEQ ID NO:7) or Asn-X-Thr-X (SEQ ID NO:8), where X is any amino acid except for Pro, thereby creating an N-glycosylation site at those positions. Creating a glycosylation site at the above described position(s) would be expected to result in a molecule that is less immunogenic in humans than the unmodified molecule. Nucleic acids encoding these variant polypeptides are also contemplated herein as are vectors and host cells comprising those nucleic acids.